Biology subjects

Ugonotti, J.

Publications and source records attributed to Ugonotti, J..

3 recordsLinked to original sources

bioRxiv2023

Glycoproteome remodelling and granule-specific N-glycosylation accompany neutrophil granulopoiesis

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we perform comprehensive spatiotemporal N-glycoproteome profiling of isolated granule populations from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Interestingly, the granules of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, peculiar highly truncated N-glycosylation in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven primarily by changes in protein expression and less by the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with high glycosylation efficiencies of the azurophilic granule proteins. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring an intriguing granule-specific N-glycosylation formed by dynamic remodelling during myeloid progenitor-to-neutrophil maturation. Key pointsO_LISystems glycobiology reveals that profound N-glycoproteome remodelling accompanies early neutrophil granulopoiesis C_LIO_LIPrecision glycoproteomics produces detailed cartography of neutrophils that exhibit site-, protein- and granule-specific N-glycosylation C_LI

systems biology↗

bioRxiv2021

Oligomannosylation and MAN1A1 expression associate strongly with a subset of human cancer types

Aberrant protein glycosylation is a prominent cancer feature. While many tumour-associated glycoepitopes have been reported, advances in glycoanalytics continue to uncover new associations between glycoproteins and cancer. Guided by a comprehensive literature survey suggesting that oligomannosylation (Man5-9GlcNAc2, M5-M9) is a widespread albeit poorly studied glyco-signature in human cancers, we here re-visit a valuable compilation of nearly 500 LC-MS/MS N-glycomics datasets acquired across 11 human cancer types to systematically test for oligomannose-cancer associations. Firstly, our quantitative glycomics data obtained across 34 cancerous cell lines demonstrated that oligomannosylation, particularly the under-processed M7-M9, is a strong pan-cancer feature. We then showed cell surface expression of oligomannosidic epitopes in the promyelocytic leukemic HL-60 cell line using concanavalin A-based flow cytometry. In keeping with literature, our quantitative glycomics data of tumour and matching control tissues and new MALDI-MS imaging data of tissue microarrays showed a strong cancer-associated elevation of oligomannosylation in both basal cell (p = 1.78 x 10-12) and squamous cell (p = 1.23 x 10-11) skin cancer and colorectal cancer (p = 8.0 x 10-4). The glycomics data also indicated that few cancer types including gastric and liver cancer exhibit unchanged or reduced oligomannose levels, observations also supported by literature and MALDI-MSI. Finally, data from cancer repositories indicated that three 1,2-mannosidases dictate oligomannose expression in cancer cells, and further suggested that deleterious mutations and reduced expression of MAN1A1 are key contributors to the cancer-associated oligomannose elevation. Collectively, these findings open hitherto unexplored avenues for the development of new cancer biomarkers and therapeutic targets.

biochemistry↗

bioRxiv2020

Hyper-Truncated N-Acetyl-β-D-Glucosamine Decorations Augment the Activity and Inhibition Potential of Neutrophil Granule Myeloperoxidase

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, structural modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity. SignificanceMyeloperoxidase (MPO) is an important microbicidal glycoprotein critical for fighting pathogens. We report, for the first time, the intriguingly complex relationship between glycobiology and MPO immune function by demonstrating that uncommon and strategically positioned hyper-truncated glycans both elevate the activity and the inhibition potential of this pathogen-combating enzyme. We have used a multifaceted approach employing integrated biomolecular analytics to generate new insights into the sugar code of MPO. The findings described in this study improve our understanding of key innate immune processes and may guide future glycoengineering efforts aiming to generate therapeutically relevant recombinant MPO products with tuneable activity and inhibition potential tailored to biomedical applications involving persisting and severe pathogen infections.

biochemistry↗